install.packages("vegan")
BiocManager::install("phyloseq")12 Characterizing community diversity
Learning Objectives
After completing this lab you should be able to
- transform metabarcoding results using
phyloseq - use
vegan:diversity()to calculate standard measure of diversity
You should have already downloaded the project directory for this module, make sure the directory is unzipped and move it to your bi328 directory. You can open the Rproj for this module either by double clicking on it which will launch Rstudio or by opening Rstudio and then using File > Open Project or by clicking on the Rproject icon in the top right of your program window and selecting Open Project.
Once you have opened a project you should see the project name in the top right corner1.
1 Pro tip: If you run into issues where a quarto document won’t render or file paths aren’t working (especially if things were working previously) one of your first steps should be to double check that the correct Rproj is loaded.
There should be a document named 12_community-diversity.qmd in your project directory. Use that file to work through this tutorial - you will hand in your rendered (“knitted”) quarto file as your homework assignment. So, first thing in the YAML header, change the author to your name. You will use this quarto document to record your answers.
In this case, this chapter doubles as your skills challenge for the week.
Before we start we need to install a package before we get started. vegan is a package that has a wide range of functions that implement standard
Now we can load the packages we need
# load libraries
library(phyloseq)
library(vegan)
library(knitr)
library(tidyverse)12.1 Compile data sets
We are going to import the data you will need for this chapter exploring how to characterize biological communities. We’ll start by loading to objects into your environment. One is the ASV table and the second is the corresponding taxonomy table from the fungi data set we were working on in the previous chapter.
We are going to read in a data set that contains information about the soil plots from which the fungi eDNA was isolated.
# load sample data
soil <- read_delim("data/soil.csv", delim = ";")The package phyloseq that we installed has various utility functions to deal with metabarcoding data set. It has a specific object class that allows us to store the asv table, taxonomy table, and sample meta data in different slots. Various functions can then be used to pull information from those slots. We can load the object ps directly into your environment using the following code.
# create phyloseq object
load(file = "data/ps.rdata")12.2 Data filtering and transformation
Before we can start exploring our data set we have a few steps to complete to transform it into containing the data we want in the format we want tit.
Let’s start by taking a looking at how many taxa are currently present in the data set using phyloseq::ntaxa()
ntaxa(ps)[1] 546We only want ASVs that were assigned as fungi in our reference database. We can use phyloseq::subset_taxa to filter by kingdom.
ps_fungi <- subset_taxa(ps, Kingdom == "k__Fungi")Next, we want to remove any singletons or doubletons. These are ASVs that are only represented by one or two reads - we can assume these are artifacts.
ps_fungi_nosd <- filter_taxa(ps_fungi, function(x) sum(x) > 2, TRUE)In many situations, metabarcoding eDNA samples is semi-quantitative2. The number of DNA templates in the environment are correlated to the number of individuals/biomass of a given taxonomic group. While you cannot necessarily back calculate the absolute abundance or number of specimen present in an environment you can use the proportion of reads assigned to a taxonomic group as a metric of relative abundance.
2 We can always confidentally use metbarcoding data as qualitative data, i.e. as presence/absence data. Though even here we should be careful about whether “not detected” should be interpreted as absent.
One way to convert species abundance from absolute to relative is using a Hellinger transformation which standardizes the abundances to the sample totals and then square roots them. The function phyloseq::transform_sample_counts() allows us to apply a function to transform sample counts.
ps_fungi_nosd_hel <- transform_sample_counts(ps_fungi_nosd, function(x) sqrt(x/sum(x)))To complete our data filtering and transformation we will use phyloseq::tax_glom() to collapse ASVs asigned to the same taxonomic group at the species level.
ps_transf = tax_glom(ps_fungi_nosd_hel, "Species", NArm = FALSE)Let’s explore our final transformed data set
Recall that our phyloseq object contains a slot that holds our ASVs table and our taxonomic table that we can access at as such.
otu_table(ps_transf)[1:2, 1:2]OTU Table: [2 taxa and 2 samples]
taxa are columns
ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTTTGGTATTCCGAAGGGCATGCCTGTTTGAGTGTCATTAAATTATCAACCTTGCTCGCTTTTATTAGCTTGAGTTAGGCTTGGATGTGAGGGTTTTGCTGGCTTCCTTCAGTGGATGGTCTGCTCCCTTTGAATGCATTAGCGGGATCTCTTGTGGACCGTCACTTGGTGTGATAATTATCTATGCCTTGAGACTTTGAAACAAACTTATGAGAATCTGCTTATAACCGTCCTCACGGACAACTTTTGACAATTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAA
S10 0.00000000
S11 0.08178608
ATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACACCTAACCTTTGGGTTTATGGCGTGGAATTGGAATGCGCCGACTGTCATGGTTGGCCCTTCTAAAATGTAGTTCTTGGCTGTCACCTAATACAGCAGTTTGGCCTAATAGTTTTGGCATTCATTGTCAAATCTTTGGCTAACATTTGCTCCAGGAGTCAGTCTTGATAATACAGAAAACTCATTCAAATTTTGATCTGAAATCAGGTAGGGCTACCCGCTGAACTTAA
S10 0.0000000
S11 0.2085144tax_table(ps_transf)[1:2, 1:2]Taxonomy Table: [2 taxa by 2 taxonomic ranks]:
Kingdom
ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTTTGGTATTCCGAAGGGCATGCCTGTTTGAGTGTCATTAAATTATCAACCTTGCTCGCTTTTATTAGCTTGAGTTAGGCTTGGATGTGAGGGTTTTGCTGGCTTCCTTCAGTGGATGGTCTGCTCCCTTTGAATGCATTAGCGGGATCTCTTGTGGACCGTCACTTGGTGTGATAATTATCTATGCCTTGAGACTTTGAAACAAACTTATGAGAATCTGCTTATAACCGTCCTCACGGACAACTTTTGACAATTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAA "k__Fungi"
ATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACACCTAACCTTTGGGTTTATGGCGTGGAATTGGAATGCGCCGACTGTCATGGTTGGCCCTTCTAAAATGTAGTTCTTGGCTGTCACCTAATACAGCAGTTTGGCCTAATAGTTTTGGCATTCATTGTCAAATCTTTGGCTAACATTTGCTCCAGGAGTCAGTCTTGATAATACAGAAAACTCATTCAAATTTTGATCTGAAATCAGGTAGGGCTACCCGCTGAACTTAA "k__Fungi"
Phylum
ATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTTTGGTATTCCGAAGGGCATGCCTGTTTGAGTGTCATTAAATTATCAACCTTGCTCGCTTTTATTAGCTTGAGTTAGGCTTGGATGTGAGGGTTTTGCTGGCTTCCTTCAGTGGATGGTCTGCTCCCTTTGAATGCATTAGCGGGATCTCTTGTGGACCGTCACTTGGTGTGATAATTATCTATGCCTTGAGACTTTGAAACAAACTTATGAGAATCTGCTTATAACCGTCCTCACGGACAACTTTTGACAATTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAA "p__Basidiomycota"
ATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACACCTAACCTTTGGGTTTATGGCGTGGAATTGGAATGCGCCGACTGTCATGGTTGGCCCTTCTAAAATGTAGTTCTTGGCTGTCACCTAATACAGCAGTTTGGCCTAATAGTTTTGGCATTCATTGTCAAATCTTTGGCTAACATTTGCTCCAGGAGTCAGTCTTGATAATACAGAAAACTCATTCAAATTTTGATCTGAAATCAGGTAGGGCTACCCGCTGAACTTAA "p__Mucoromycota" 12.3 Combine data sets
We have three explanatory variables that could be driving differences in fungal communities among samples.
- Soil samples were taken in differnt forest plots that were classified as dominated by Acer saccharum (AS) or Fagus grandifolia (FG) or mixed with other small trees and shrubs present (mixed).
- Soil samples where taken from different soil horizons (depths): L, F, H, Ae, or B
- Soil chemistry (carbon, nitrogen, pH)
This information is stored in our soil dataframe.
Now that we’ve filtered and transformed our data set, let’s pull it back out to create a dataframe as the object you are more familiar with in terms of being able to manipulate it.
12.4 Characterize community diversity
Okay… now we’re ready to have some fun.
First, let’s find out what taxonomic groups are present in the entire data set.
3 use the function kable() to print the entire data frame in a pretty table
Measures of diversity enable us to quantify the complexity of biological communities in a way that allows us to objectively compare communities across space and time. Measures of alpha diversity describe the diversity of a single sample and are based on the number of observed taxonomic groups or their relative abundance, beta diversity describes the variation between samples, gamma diversity describes the global diversity observed across a large number of communities.
Common alpha diversity statistics include:
- observed richness the number of observed taxa.
- Shannon Index (Shannon-Weaver or Shannon-Wiener Index) quantifies how difficult it is to predict the identity of a randomly sampled individual. Ranges from 0 (total certainty) to log(S) (total uncertainty).
- Simpson Index quantifies the probability that two randomly chosen individuals are the same taxonomic group (ranges from 0 to 1).
- Inverse Simpson Index is defined as the number of species needed to have the same Simpson index value as the observed community assuming a theoretical community where all species are equally abundant.
The Shannon and Simpson Diversity are entropy-based indices that measure the disorder (diversity) of a system. In information theory entropy is used to describe the fact that we can quantify the degree of uncertainty associated with with predicted pieces of information. Applied to ecology this means that when describing diversity using these indices we are determining whether or not individuals randomly drawn from a community are the same or different species (or other taxonomic group).
The relationship between species richness and Shannon diversity is non-linear, i.e. at higher levels of species richness, communities appear more similar in terms of the magnitude of the index compared to lower levels of species richness - which is counter intuitive to the way species richness works. The solution to this is to linearize the indices. As a result, more recently, diversity indices have been proposed where diversity values are converted into equivalent (or effective numbers) of species (jost_entropy_2006?). The effective number of species is the number of species in a theoretical community where all species (taxonomic groups) are equally abundant that would produce the same observed value of diversity (a similar principle is applies in genetics for the concept of effective population sizes). While the definition of effective numbers is not as intuitive as the entropy-based ones the values that we yield are. Not only are the “units” species (instead of being a unitless index), but they have properties that we intuitively understand. For example, effective numbers obey the doubling principle: If you have two communities with equally abundant but totally distinct species and combine them, that new community would have a diversity that is 2x that of the original ones.
The package vegan has several functions implemented that allow us to calculate these diversity stats. Look up any functions you are not familiar with in the following code and comment it to describe what each line of code is doing.
diversity <- tidy_counts %>%
group_by(ID, forest, horizon) %>%
summarize(richness = specnumber(count),
shannon = diversity(count, index = "shannon"),
simpson = diversity(count, index = "simpson"),
invsimpson = diversity(count, index = "invsimpson")) %>%
pivot_longer(cols = c(richness, shannon, simpson, invsimpson),
names_to = "metric")We can create a series of plots that compare the different diversity stats for each forest plot type and soil horizon location4.
4 Well, I can but you will be able to soon, too
ggplot(diversity, aes(x = forest, y = value, color = forest)) +
geom_boxplot(aes(color = forest), outlier.shape = NA, fill = "transparent", size = 1) +
geom_point(aes(fill = forest),
position = position_jitterdodge(jitter.width = .3),
shape = 21, color = "black", size = 3) +
facet_grid(metric ~ horizon, scales = "free") +
labs(x = " ", y = " ")